PROJECT SUMMARY No effective therapeutic agents currently exist to improve outcomes in autism and related neurodevelopmental disorders. Genetic research has led to the discovery of a growing list of highly penetrant mutations that contribute to disease pathophysiology. This recent progress provides an important opportunity to define the molecular mechanisms underlying these disorders, as well as to identify targets for new treatment strategies. However, given the large number of emergent loci, a major challenge for the current phase of autism research is to establish convergent cellular mechanisms that group apparently distinct genetic etiologies. Here we focus on the following disease-associated proteins: the E3 ubiquitin ligase UBE3A/E6-AP and Na+/H+ exchangers (NHEs), with an emphasis on NHE6 (also known as SLC9A6). Genetic mutations affecting these proteins are associated with overlapping clinical syndromes. These shared clinical spectra suggest that UBE3A and NHEs may function in a convergent cellular pathway. Exciting preliminary data support a functional interaction between UBE3A and NHE6 in the regulation of intra-organellar pH. Discovery of a shared pathway for UBE3A and NHEs involving intra-organellar pH would be fortuitous because FDA-approved drugs known to target this cellular process are currently available. Therefore, success in this research would lay the foundation for future drug screening in available models of disease, including both patient-derived iPSCs and animal models. Our central hypothesis is that UBE3A controls trafficking and degradation of NHEs, and that perturbations in UBE3A activity (through loss-of-function or gain-of-function mutations) cause abnormalities in the regulation of intra-organellar pH as a result of mislocalization and aberrant accumulation of NHEs. Such abnormalities in intra-organellar pH will disturb organellar functions, including protein processing, trafficking, and signaling, and ultimately disrupt circuit development. We will test this hypothesis and build a path for future research through these Specific Aims: (1) Determine the extent to which UBE3A governs the trafficking and degradation of NHE proteins; and (2) Investigate perturbations in regulation of intra-organellar pH mediated by UBE3A and NHE6. We are conducting innovative, live-cell imaging of intra-organellar pH in neurons, from both patient-derived iPSCs and also animal models, by targeting ratiometric pHluorin to specific organellar compartments. The research is significant because: (1) The discovery of a functional linkage between autism-associated genes serves the important goal of developing more unified pathogenic mechanisms yielding treatment targets; and (2) UBE3A has been a widely studied protein; however, a role in modulation of intra-organellar pH represents a new mechanism with a potential for targeting by therapeutic drugs.